Purification of Monoclonal Antibodies from Cell-Culture Supernatant by Use of Anion-Exchange Convective Interaction Media (CIM) Monolithic Columns

Dhivya, Ambur P.; Kumar, B. Prem; Prasanna, Rajasekar R.; Jayaprakash, N.S.; Vijayalakshmi, M.A.

doi:10.1365/s10337-010-1787-3

Cited by 7 publications

(8 citation statements)

References 23 publications

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“…The high selectivity and extension to a wide variety of ligand chemistry with CIM media have gained attention in the area of bio separation [35]. There are already several reports on purification of macromolecules like DNA, RNA, viruses like particles, IgM, IgG and enzymes using CIM ion exchange systems [35][36][37][38][39][40]. In our study, we used a CIM-CM disk for the purification of uricase enzyme.…”

Section: Discussionmentioning

confidence: 99%

Purification and characterization of an extracellular uricase from a new isolate of Sphingobacterium thalpophilum (VITPCB5)

Ravichandran

Hemaasri

Cameotra

et al. 2015

Protein Expression and Purification

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Section: Discussionmentioning

confidence: 99%

Purification and characterization of an extracellular uricase from a new isolate of Sphingobacterium thalpophilum (VITPCB5)

Ravichandran

Hemaasri

Cameotra

et al. 2015

Protein Expression and Purification

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“…The retained and nonretained proteins from each chromatographic experiments were analyzed for antigen binding efficiency by an antigen specific ELISA following the method of Dhivya et al (2010). Human serum albumin (1 mg/well) was used as coating antigen.…”

Section: Antigen Binding Efficiencymentioning

confidence: 99%

“…The fast method developed on the laboratory scale can be subsequently efficiently transferred to a preparative scale and more importantly to the industrial scale (Barut et al, 2008;Jungbauer and Hahn, 2008;Kramberger et al, 2010;Podgornik et al, 2004). CIM disks with different ligands have been used for antibody purification from sera (Berruex et al, 2000;Ostryanina et al, 2002;Brne et al, 2006), mouse ascites and cell culture supernatant (Champagne et al, 2007;Dhivya et al, 2010).…”

Section: Introductionmentioning

confidence: 99%

Purification of monoclonal antibodies, IgG1, from cell culture supernatant by use of metal chelate convective interaction media monolithic columns

Rajak

Vijayalakshmi

Jayaprakash

2012

Biomedical Chromatography

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Monoclonal antibodies (MAbs) have diverse applications in diagnostics and therapeutics. The recent advancement in hybridoma technology for large-scale production of MAbs in bioreactors demands rapid and efficient purification methods. Conventional affinity purification systems have drawbacks of low flow rates and denaturation of antibodies owing to harsh elution conditions. Here, we attempted purification of MAbs by use of a high-throughput metal-chelate methacrylate monolithic system. Monolithic macroporous convective interaction media-iminodiacetate (CIM-IDA) disks immobilized with four different metal ions (Cu²⁺, Ni²⁺, Zn²⁺ and Co²⁺) were used and evaluated for purification of anti-human serum albumin IgG1 mouse MAbs from cell culture supernatant after precipitation with 50% ammonium sulfate. Elution with 10 mM imidazole in the equilibration buffer (25 mM MMA = MOPS (Morpholino propane sulfonic acid) + MES (Morpholino ethane sulfonic acid) + Acetate + 0.5 M NaCl, pH 7.4) resulted in a purification of 25.7 ± 2.9-fold and 32.5 ± 2.6-fold in experiments done using Zn²⁺ and Co²⁺ metal ions, respectively. The highest recovery of 85.4 ± 1.0% was obtained with a CIM-IDA-Zn(II) column. SDS-PAGE, ELISA and immuno-blot showed that the antibodies recovered were pure, with high antigen-binding efficiency. Thus, metal chelate CIM monoliths could be a potential alternative to conventional systems for fast and efficient purification of MAbs from the complex cell culture supernatant.

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“…ELISA Serum antibody was detected by ELISA as previously described [5]. Briefly, polystyrene 96-well assay plates were coated with the antigens diluted in 0.05 M carbonate buffer at pH 9.6 (10 lg mL -1 ), overnight at 4°C [15].…”

Section: Sds-page Analysismentioning

confidence: 99%

“…Routinely, serum IgG is purified by a procedure consisting of saturated ammonium sulfate precipitation (SASP) and a subsequent chromatography such as affinity chromatography [4] or ion-exchange chromatography (IEC) [5]. As an efficient approach, IEC is widely used to purify IgG by bind-elute mode IEC (Be-IEC) or flow-through mode IEC (Ft-IEC), depending on the impurity profiles in the inlet feed stream [6,7].…”

Section: Introductionmentioning

confidence: 99%

Purification of IgG from Sera of Rabbit and Guinea Pig by Flow-Through Mode Ion-Exchange Chromatography using DEAE Sepharose Fast Flow Column

et al. 2011

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A one-step purification process using flowthrough mode ion-exchange chromatography (Ft-IEC) was evaluated for the purification of Immunoglobulin G (IgG) from rabbit and guinea pig serum. A simple protein precipitation with saturated ammonium sulfate was used to pretreated plasma samples. Chromatographic separation was carried out on DEAE Sepharose Fast Flow (F.F.) column at a flow rate of 1 mL min -1 . Ft-IEC using TrisHCl buffer at pH 7.0 allowed the recovery of 22% of the loaded IgG with purity of 95% existed in flowthrough and washing effluents but not in elution effluents. To be compared, bind-elute mode IEC using Tris-HCl buffer at pH 8.5 based on the routine protocol showed that the recovery of 15% of the loaded IgG with purity of 80% existed in elution effluents. These results indicate that the Ft-IEC is an effective method for purifying IgG from sera of rabbit and guinea pig and may also be suitable for other animal sera by adjusting pH at equilibrium buffer for chromatography column.

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Purification of Monoclonal Antibodies from Cell-Culture Supernatant by Use of Anion-Exchange Convective Interaction Media (CIM) Monolithic Columns

Cited by 7 publications

References 23 publications

Purification and characterization of an extracellular uricase from a new isolate of Sphingobacterium thalpophilum (VITPCB5)

Purification and characterization of an extracellular uricase from a new isolate of Sphingobacterium thalpophilum (VITPCB5)

Purification of monoclonal antibodies, IgG1, from cell culture supernatant by use of metal chelate convective interaction media monolithic columns

Purification of IgG from Sera of Rabbit and Guinea Pig by Flow-Through Mode Ion-Exchange Chromatography using DEAE Sepharose Fast Flow Column

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